This proposal is designed to test the overall hypothesis that reconstitution of C18-ceramide generation mediates lethal autophagy, subsequently leading to mitochondrial dysfunction, and suppression of HNSCC tumor growth. As a corollary, the hypothesis that treatment of HNSCCs with C18-ceramide analogues, such as C18-pyridinium-ceramide (C18-Pyr-Cer), which enhance LC3-lipidation and mitochondrial targeting, induces autophagy-mediated tumor suppression will also be tested. To test these novel hypotheses, three Specific Aims are proposed: Specific Aim 1. Determine the roles of CerS1-generated C18-(dihydro)ceramide in the regulation of HNSCC growth inhibition via induction of autophagy. Specific Aim 2. Define the down-stream mechanisms by which CerS1/C18-ceramide signaling induces lethal autophagy. Specific Aim 3. Define the roles of C18-ceramide and LC3-II in selective mitochondrial targeting of autophagosomes to enhance lethal autophagy and tumor suppression. Collectively, studies proposed in this application will help define novel mechanisms involved selectively in the regulation of lethal autophagy by C18-ceramide signaling, which subsequently leads to suppression of HNSCC tumor growth. PUBLIC HEALTH RELEVANCE: Because novel strategies are needed for the treatment of human head and neck squamous cell carcinoma (HNSCC), the long-term objective of this proposal is to develop mechanism based therapeutic strategies for the treatment of this disease. To this end, these proposed studies will identify novel mechanisms by which C18- ceramide selectively induces lethal autophagy, leading to the suppression of HNSCC tumor growth. Data obtained from these studies will also help resolve the autophagy paradox, and define checkpoint mechanisms involved in inducing lethal versus survival autophagy by ceramide signaling, which will lead to the development of novel therapeutics against HNSCC.